Acidified nitrite as an antimicrobial agent

ABSTRACT

The present invention provides a dosage form for the treatment of bacterial, viral or fungal conditions which comprises, a pharmaceutically acceptable acidifying agent in an amount sufficient to reduce the pH at an environment of use to below pH4, and a pharmaceutically acceptable source of nitrite ions or a nitrate precursor therefor; wherein said acidifying agent and said source of nitrite ions or nitrate precursor are separately disposed in respective pharmaceutically acceptable carriers for admixture at the intended environment of use to release NO or NO 2  ions.  
     The invention also provides delivery systems for the topical medicament.

[0001] The present invention relates in one aspect to acidified nitriteas an antimicrobial agent, and to a complex of nitrogen oxides arisingfrom the interaction of nitrite and acid as an antiviral composition forthe treatment of viral diseases of the skin by topical applicationthereto. Such nitrogen oxides include in particular NO which is ofimportance particularly if acidified.

[0002] An active entero-salivary circulation in man provides acontinuous flow of nitrate into the mouth where it is rapidly reduced tonitrite by bacteria on the tongue. The effect of salivary nitrateexcretion is to provide a precursor for the generation of nitrogenoxides by the break down of the nitrite.

[0003] In brief we have found that exposure of a yeast, Candida albicansand the bacterium E coli to concentrations of nitrite in saliva togetherwith acid conditions similar to those found in the stomach for one hourcaused a dose-dependent reduction in their survival. It is apparenttherefore that the generation of nitrogen oxides and/or nitrous acid inthe mouth and in the gastrointestinal tract, particularly the uppergastrointestinal tract, from acidified nitrite is preventative ofmicrobial infection.

[0004] In the mouth bacteria rapidly reduce nitrates to nitrites. Onceswallowed the acid conditions of the stomach protonate the nitrite toform nitrous acid (pKa approx 3.5). The nitrous acid in turn dissociatesto form oxides of nitrogen as shown below.

NO₂ ⁻+H+═HNO₂   (1)

2HNO₂═H₂O+N₂O₃   (2)

N₂O₃═NO+NO₂   (3)

N₂O₃+C₂H₈O₆=2NO+H₂O+C₆H₆O₆   (4)

[0005] Endogenous and dietary nitrate is actively concentrated bysalivary glands to more than 10 times the concentration in plasma andsecreted in saliva. Thus the saliva provides a continuous source ofnitrate to the upper gastrointestinal tract. Oral conversion of nitrateto nitrite is rapid and is restricted to the surface of the tongue inman and to the posterior third of the tongue in the rat.

[0006] The function of the entero-salivary circulation of nitrate is notknown but it may well be that gastric acid by itself is not alwayssufficient to destroy many ingested micro-organisms and that the primaryrole of salivary nitrate secretion and conversion to nitrite is as aprecursor for nitrogen oxides in the lumen of the stomach which willkill swallowed micro-organisms.

[0007] The above identified mechanism is also applicable to thedestruction of micro-organisms on an in the skin. For example athlete'sfoot or tidea pedis.

[0008] In WO 95/22335 we have disclosed a pharmaceutical compositioncomprising a pharmaceutically acceptable source of nitrites and apharmaceutically acceptable acidifying agent, inter alia for the directtreatment of disease by topical application. These compounds have adirect effect on the organism concerned but the precise mode of actionis not known.

[0009] U.S. Pat. No. 4,595,591 reveals a composition comprising anaqueous solution of nitric acid and nitrous acid at a pH below 1preferably with a organic acid and copper and cadmium ions for thetreatment of superficial lesion of the skin, for example tumorousgrowths.

[0010] U.S. Pat. No. 5,648,101 provides a vaso-active compositioncomprising NO adapted for delivery to a body site inter alia by means ofa cream or ointment. The NO is generated from an admixture of ferroussulphate, an organic acid and an inorganic nitrite and caused to bereactive in the presence of moisture adjacent or at the site.Acidification is not discussed.

[0011] WO 96/02268 reveals the inhibition of a virus by nitric oxide(NO₂) derived from a complex unstable organic molecule, but theadvantages of reduction of pH at the environment of use have not beenappreciated, neither have the beneficial effects the chemical release ofthe NO and NO₂ moieties immediately adjacent to the environment of use,been realized.

[0012] WO 93/25213 reveals a composition comprising nitrous oxidecontained in a dermatological composition comprising as an essentialfeature a fatty acid or a lower alkyl ester thereof, pH values,particularly at the environment of use, are not mentioned.

[0013] All are single formulations which are admixed well prior toapplication to the environment of use so that NO and NO₂ all escapeprior to use and hence have a very limited utility.

[0014] We have now found inter alia that nitrite at concentrations of upto 4% in an inert carrier cream or ointment when mixed with an organicacid such as salicylic acid reacts to produce oxides of nitrogen whichare effective in killing infectious organisms on the skin includingfungi, yeast, bacteria and viruses. The combination of nitrite and acidcauses mild erythema (redness) of the skin due to release of nitricoxides at the environment of use but this causes no significantinflammation.

[0015] We have also found that as far as viruses, as opposed to bacteriafor example, are concerned, that the above nitrogen oxide complex,comprising for example NO and/or NO₂ while it may effect replication toa degree, more importantly modifies the virally infected cells such thatthe immune system can recognize the viral particles. Inter alia, this issupported by the fact that the complex is less effective inimmunosuppressed hosts. Generally the greater the percent of nitricoxide (NO) the better the immuno-potentiation. If possible up to 100% NOcan be used.

[0016] It is thought, although more work is required, that smallermolecules, particularly NO and NO₂ penetrate the skin by directdiffusion or via the seat glands or hair follicles through the epidermisto the sweat cells. It has been found that although the healthykeratinocytes find the oxides of nitrogen toxic they do not die as theyare relatively resistant to its effects. However, the surprisingclinical results in our examples lead us to believe that virallyinfected cells are more susceptible to these effects, leading todestruction of the virally infected cells via a combination of toxicityleading to programmed cell death and potentiation of the immune responseto the presence of the virus.

[0017] The above identified mechanism is also useful in thesterilisation of objects such as dentures by utilising a sterilizingnitrate solution. Conventional solutions which are effective insterilising dentures often taste unpleasant due to chlorine-baseddisinfectants. A combination of nitrite and acid results in aantimicrobial solution which has little or no taste. Other objects suchas contact lenses may be sterilised in the same way.

[0018] Gastroenteritis continues to be a major problem in rearing pigsand other farm animals. Enteropathogenic Escherichia coli (especiallythose bearing the K88 antigen) are particularly implicated. Althoughgastric acidity is thought to be one of the main host defence systemswhich provides a barrier to orally-acquired infection, this is clearlyineffective in preventing organisms from reaching the more distalintestine in these animals.

[0019] The role of NO as a compound which inhibits viral replication invitro has been disclosed by J. B. Mannick; 63^(rd) Forum in Immunology,and papers in Intervirology 1995; 38: 206-213, Trends in Microbiology1995; 3: 81-82, Science 1993; 261: 1445-1448, and The Journal ofClinical Investigation 1993; 91: 2446-2452. The above papers disclosethe effects of NO on various viruses, for example herpes simplex virus,vaccinia virus and vesicular stomatitis virus. Exogenous NO donors suchas S-nitroso-N-acetyl penicillamine (SNAP) or SIN-1 were used in vitroto determine the role of NO as an antiviral compound. Application ofexogenous NO to cell-lines infected with the virus under test resultedin inhibition of the viral DNA replication. The exact mechanism of theinhibition seemed to differ depending on the virus involved. For examplein the case of vaccinia virus it is thought that the NO may inhibitreplication by binding to non-haem iron or thiol groups that areessential for the catalytic activity of enzymes involved in vacciniareplication. In this in vitro model the antiviral effects of NO do notrequire immune recognition of infected cells thus providing an earlydefence against viral pathogens prior to the development of a specificimmune response.

[0020] In order for viruses to survive and reproduce they must evaderecognition by the hosts immune responses. The mechanism by which thisis achieved is largely unknown but an effective immune responseeradicates the infection. Viruses are obligate intracellular pathogens.They reproduce using the host's metabolic machinery.

[0021] At present drug treatment of viral diseases is predicted upon asmall number of compounds which block the replication of the virus. Forexample Acyclovir, which is effective against herpes virus, is adeoxyguanosine analogue which competes with deoxyguanosine triphosphateas a substrate for viral thymidine kinase and when phosphorylated andincorporated in the viral DNA causes premature DNA chain termination.

[0022] Unfortunately anti-viral drugs are only effective for a limitednumber of viral infections and viruses can mutate to overcome theeffectiveness of the drugs. In the case of molluscum contagiosum 1 and2, which are related to orthopox and parapox viruses and share somehomology with vaccinia, other forms of treatment have to be used.Current therapies comprise physical destruction with manual extrusion,liquid nitrogen therapy or curettage, all of which are painful and notvery effective and may cause scarring. The pain of these therapies isparticularly pertinent because the majority of patients are under theage of 10 years.

[0023] In the case of recalcitrant warts, destructive therapies such asliquid nitrogen can be used in cases where the conventional salicylicacid paints have not resulted in the warts disappearance. One problemwith warts is that the viral pool is in the stem cells which are foundat the base of the epidermis. The aforementioned treatments often removethe virus particles and thus the infection from the top layer of theepidermis, but they do not penetrate deep enough to remove the stemcells and therefore the origins of the infection. This can result in there-emergence of the warts.

[0024] An alternative treatment for warts is by use ofdinitrochlorobenzene. Such treatment is intended to make the patientallergic to dinitrochlorobenzene, whereupon the patient's immune systemmounts an immune response to the dinitrochlorobenzene at the site of thewart and the wart in some cases disappears, presumably as a result ofimmuno-potentiation. Immuno-potentiation can be an effective treatmentbut subjecting the patient to an allergic reaction caused bydinitrochlorobenzene can be hazardous, variable and difficult tocontrol.

[0025] According therefore to a first aspect of the present inventionthere is provided a dosage form for the treatment of bacterial, virus,or fungal conditions in the human or animal body which comprises:—

[0026] a pharmaceutically acceptable acidifying agent in an amountsufficient to reduce the pH at an environment of use to below pH4, and

[0027] a pharmaceutically acceptable source of nitrite ions or a nitrateprecursor therefor;

[0028] wherein said acidifying agent and said source of nitrite ions ornitrate precursor are separately disposed in respective pharmaceuticallyacceptable carriers for admixture at the intended environment of use torelease NO or NO₂ ions.

[0029] Preferably the acidifying agent is an organic acid, for examplesalicylic acid or ascorbic acid. The precursor for the NO₂ or NO moietymay be an alkaline metal or alkaline earth metal nitrate capable ofconversion to NO₂ or NO by enzymic action.

[0030] The pharmaceutical acceptable carrier or diluent may be an inertcream or ointment. In a particularly preferred form of the invention theacidifying agent and the source of nitrite ions or precursor thereforare separately disposed in said cream or ointment for admixture torelease NO₂ or NO ions at the environment of use. Alternatively an acidcomposition may be presented for administration in tablet or liquidform.

[0031] Depending on the type of viral infection the components of thenitrogen oxide can work synergistically or alone. Nitrogen oxides, forexample NO and NO₂, particularly can diffuse through the epidermis. Inthe case of warts this allows them to reach the stem cells which are atthe base of the epidermis and are the cells which contain the pool ofestablished virus. Once at the infected cells the nitrogen oxide complexcan facilitate programmed cell death, selectively in infected cells,which may then be taken up by phagocytes and antigen presenting cellsleading to immune recognition of the previously hidden viral antigens.Once recognized, specific immunity will lead to destruction of allinfected cells through cellular and humoural responses.

[0032] Accordingly therefore to a further aspect of this invention thereis provided a method of exposing virally infected cells at or adjacentan environment of use to a mammalian immune response in vivo, whichcomprises applying to said cells an admixture of nitrogen oxidesgenerated by admixing at the environment of use a pharmaceuticallyacceptable acidifying agent in an amount sufficient to reduce the pH atthe environment of use to below pH4 and a pharmaceutically acceptablesource of nitrogen oxides or a nitrate precursor therefor.

[0033] According to a second aspect of the invention there is providedthe use in the manufacture of a topical medicament for the in vivopotentiation of the immune system during a viral skin infectionresultant from virus replication in the epidermis, of topicalformulations comprising a separately disposed source of pharmaceuticallyacceptable nitrogen oxides, and a separately disposed pharmaceuticallyacceptable acidifying agent, for admixture, at an intended environmentof use to release NO and NO₂moieties.

[0034] Preferably the viruses replicating in the epidermis which causethe viral skin infection are selected from molluscum contagiosum, herpessimplex type 1 and 2, varicella zoster virus and papilloma virus.Treatment using the acidified nitrogen oxide source has been shown to beparticularly effective in viral skin infections caused by theaforementioned viruses.

[0035] Conveniently the source of nitrogen oxides contains nitric oxideand may also contain NO− or NO+ nitrosium ions or a precursor thereforproduced when a pharmaceutically acceptable acidifying agent and apharmaceutically acceptable donor of nitrogen oxides or a precursortherefor, are brought into intimate contact at a site of biologicalaction (environment of use).

[0036] If the pharmaceutically acceptable acidifying agent and thepharmaceutically acceptable donor of nitrogen oxides, or a precursortherefor were brought into contact before reaching the site ofbiological action the efficacy of the treatment is diminished as thenitrogen oxides become progressively more inactive with time.

[0037] In a preferred embodiment the pharmaceutically acceptableacidifying agent, the pharmaceutically acceptable donor nitrogen oxidesor a precursor therefor are each separately disposed in apharmaceutically acceptable carrier or diluent.

[0038] Preferably the pharmaceutically acceptable acidifying agent is anorganic acid or salt with a low pH such as ascorbyl palmitate. Theorganic acid may be selected from at least one of ascorbic acid,ascorbyl palmitate, salicylic acid, lactic acid, citric acid, formicacid, benzoic acid and tartaric acid.

[0039] The choice of acidifying agent depends on the type of infectionof the skin and the reaction of the infected areas to treatment. The useof reducing acids such as ascorbic acid gives a quick burst of NO andNO₂ with significantly more NO produced compared to the amount of NO₂produced. The other organic acids such as salicylic acid give asustained concentration of NO and NO₂ over a certain time period whereinthe ratio of NO to NO₂ is low. The concentration of the inorganicnitrite, for example sodium nitrite (or other alkali metal nitrites), asthe pharmaceutically acceptable donor of nitrogen oxides or a precursortherefor depends on the acid used and the concentration of the acidused. The reducing acid ascorbic acid is highly reactive so thereforeonly between 1-10% is required with stoichiometric concentrations of thepharmaceutically acceptable donor of nitrogen oxides or a precursortherefor (e.g. sodium or other alkali metal nitrite). Ascorbyl palmitateis more stable but requires a higher concentration than ascorbic acidbecause the palmitate has a higher molecular weight. A concentration ofbetween 3% and 25% of ascorbyl palmitate is thus required. If salicylicacid is used, concentrations of between 0.5% and 30% are appropriate,citric acid requires a yet higher concentration of up to 45%. (All %given herein are by weight).

[0040] The concentration of sodium nitrite required to react with theabove mentioned concentrations of organic acid is between 0.5% and 30%,preferably between 5% and 20%. Other pharmaceutically acceptable sourcesof nitrogen oxides or a precursor therefor require different ranges ofconcentration.

[0041] Preferably the pharmaceutically acceptable acidifying agent andthe pharmaceutically acceptable donor of nitrogen oxides or a precursortherefor are in stoichiometric concentrations. If the pharmaceuticallyacceptable acidifying agent and the pharmaceutically acceptable donor ofnitrogen oxides or a precursor therefor are in stoichiometricconcentrations, after the reaction is completed there will be nounreacted compounds left. Accordingly any compounds remaining on theinfected area will not be able to contaminate healthy skin with theactive medicament or anything the treated area touches such as furnitureand clothing.

[0042] In a preferred embodiment the medicament is in the form of apaint, a varnish, an ointment, a wax, a salve, or a cream. Theseembodiments allow the pharmaceutically acceptable acidifying agent and apharmaceutically acceptable donor of nitrogen oxides, or a precursortherefor to be applied directly to the infected area. The treatmentcomprising the topical application of separate compositions according tothis invention is preferably continued for at least one month, and morepreferably two months.

[0043] In a further aspect of the present invention there is provided atwo-part delivery system for the topical application of a medicament forthe in vivo treatment of the epidermis, the said system comprisingseparately;

[0044] a first waxy component comprising a pharmaceutically acceptableacidifying agent;

[0045] and a second waxy component comprising a pharmaceuticallyacceptable source of nitrogen oxides whereby if topically applied invivo simultaneously, or immediately sequentially, to the environment ofuse, active nitrogen oxides are released therefrom.

[0046] In a further embodiment, the first and second waxy componentscomprise a paraffin. The acidifying agent is preferably a reducingorganic acid or salt such as ascorbic acid or ascorbyl palmitate. Thesource of nitrogen oxides may be an alkali metal nitrite such as sodiumnitrite.

[0047] The use of a reducing acid or salt thereof results in a productreleased at the environment of use which comprises a major amount of NOwhich has significant therapeutic and immunological effects.

[0048] Thus the invention provides for the use of a source of oxide(s)of nitrogen in the manufacture of a composition for the treatment orprophylaxis of a viral skin infection by a virus selected from herpessimplex types 1 and 2, varicella zoster, vaccinia or papilloma, andparticularly from molluscum contagiosum.

[0049] In a further aspect of the invention there is provided a deliverysystem for the topical application of a medicament for the in vivotreatment of the epidermis, comprising an adhesive layer and a supportlayer impregnated with at least one of the components of the medicament,characterized in that the components of the medicament are apharmaceutically acceptable acidifying agent and a pharmaceuticallyacceptable donor of nitrogen oxides or a precursor therefor, and a meansof combining the pharmaceutically acceptable acidifying agent with thedonor of nitrogen oxides. For example the delivery system may comprisetwo layers, which when in situ release the oxides of nitrogen includingnitric oxide. The activation can be by pressure applied or by hydrationfrom the skin.

[0050] Preferably the delivery system is adapted for the potentiation ofthe immune system during a viral skin infection resultant from virusreplication with the delivery system in place, such a system may, forexample, resemble an adhesive plaster so it is then simple to applyphysical pressure to the exterior of the plaster.

[0051] Conveniently the donor of pharmaceutically acceptable nitrogenoxides may be aqueous and encapsulated in microspheres or liposomesdisposed in the support, preferably in the form of a film or a gauze.The film or gauze allows increased concentrations of thepharmaceutically acceptable acidifying agent to be used. If a solutionof salicylic acid is used then only a concentration of 20-26% by weightis applied, but if salicylic acid is impregnated in the film or thegauze then a concentration of 26 to 44% by weight can be applied.

[0052] A further advantage of using an adhesive layer is that it can beused to occlude the infected area during treatment which increases theconcentration of nitrogen oxides being absorbed through the epidermis.

[0053] Another advantage of using the delivery system as just described,instead of two creams or ointments, is that the components of themedicament will only be applied to the infected site, i.e. no spillagewill occur. It is also easier for the elderly, who may have shaky hands,to apply the adhesive layer rather than applying a paint. For thetreatment of molluscum contagiosum, which is mainly found in those underthe age of 10 years, the adhesive layer can be a decoratively patternedin order to appeal to children.

[0054] As stated above preferably the integrity of the vehicle isdisrupted by pressure after application of the adhesive layer and filmor gauze to a site of viral infected skin. If the pharmaceuticallyacceptable acidifying agent and the pharmaceutically acceptable nitrogenoxide donors or precursors therefor are not kept separate untiladministration at the site of biological action they will react togetherthus rendering the medicament less effective. Accordingly, in thisembodiment it is necessary for the pharmaceutically acceptableacidifying agent and the pharmaceutically acceptable nitrites orprecursors therefor to be retained separately within the film or gauzelayer. The application to the site of biological action of pressureapplied to the adhesive layer, and therefore the film or gauze layer,can result in the vehicles, such as the microspheres or lipsomes,breaking and the pharmaceutically acceptable acidifying agent and thepharmaceutically acceptable nitrogen oxide donors or precursors thereforreacting, thus treating the infected area.

[0055] In another aspect the delivery system may be used in conjunctionwith a topically applied medicament. The topically applied medicamentbeing either a pharmaceutically acceptable acidifying agent or apharmaceutically acceptable donor of nitrogen oxides or a precursortherefor.

[0056] It is thus possible to provide only one of either thepharmaceutically acceptable acidifying agent or the pharmaceuticallyacceptable nitrogen oxide donors or precursors therefor impregnated inthe film or gauze layer. The other compound, which is not impregnated inthe film or gauze can then be applied topically to the infected site.The advantage of this arrangement is that the film or gauze layer can belarger than the infected site but a reaction between thepharmaceutically acceptable acidifying agent and the pharmaceuticallyacceptable nitrogen oxide donors or precursors therefor only occurs atthe infected site where the medicament had been topically applied.

[0057] It is also possible to vary the treatment regime by changing thetopically applied medicament without changing the compound in thedelivery system. For example if the pharmaceutically acceptable nitrogenoxide donors or precursors therefor are impregnated in the film orgauze, then the type of pharmaceutically acceptable acidifying agentthat is topically applied can be altered and the same adhesive and filmor gauze layers utilized.

[0058] The delivery system is an ideal form of treatment for theverrucae on the feet because the delivery system is hidden from view.

[0059] In a further aspect of the invention there is provided a methodof sterilising an object which method comprises the steps of:

[0060] 1) preparing a pharmaceutically acceptable acidifying agent and apharmaceutically acceptable source of nitrite ions,

[0061] 2) mixing said acidifying agent with said source of nitrite ionsin a liquid carrier or diluent in contact with said object thereby toreduce the pH to below 4 while causing said sterilant nitrite ions tosterilize said object.

[0062] In a further form of the invention there is provided a sterilantcomposition comprising a pharmaceutically acceptable acidifying agent,

[0063] a pharmaceutically acceptable source of nitrite ions or a nitrateprecursor therefor,

[0064] and a pharmaceutically acceptable carrier or diluent thereforwherein the acidifying agent is adapted to reduce the pH at theenvironment of use to below pH4.

[0065] In a still further form of the invention there is provided ananimal feed supplement comprising a pharmaceutically acceptableacidifying agent and,

[0066] a pharmaceutically acceptable source of nitrite ions or a nitrateprecursor therefor, in an amount sufficient to produce a beneficialanti-microbial pharmalogical effect, but insufficient to produce adverseaction in the target animal.

[0067] The acidifying agent may be salicylic or ascorbic acid as above,and the source of nitrite ions or nitrate precursor therefor may be inan inorganic nitrate as set forth above. Where the animal is the pig,the supplement should be included in an amount sufficient to ensure thateach adult animal will receive a balanced dose of between 0.3 to 5.0g/day and preferably about 1 g/day.

[0068] The invention will now be described, by way of illustration only,with reference to the following examples and figures accompanying thespecification.

[0069]FIG. 1 shows a diagram indicative of the effect of exposure tonitrate and differing hydrogen ion concentrations on the survival of Calbicans where the vertical axis is the optical density in absorbanceunits and the horizontal axis is the pH.

[0070]FIG. 2 shows growth curves of E coli following exposure to acidalone or acid with a nitrite where the vertical axes are optical densityin absorbance units and the horizontal axes are time in hours.

[0071]FIG. 3 shows growth curves of E coli following exposure to pH3 invarious nitrite concentrations where the vertical axis shows the opticaldensity in absorbance units and the horizontal axis is time in hours.

[0072]FIG. 4 shows the generation of nitric oxide from sodium nitrite atdifferent levels of acidity where the vertical axis is the nitric oxideconcentration (nM) and the horizontal axis is Ph.

[0073]FIG. 5 shows a graph of the duration of the warts compared to thetime for wart disappearance with the formulations given in Example 7,where n=32;

[0074]FIG. 6 shows the outcome of the treatment of patients with wartsas a function of time, where n=32.

[0075]FIG. 7 shows a Kaplan Meier plot of the outcome of the treatmentof patients with molluscum contagiosum as a function of time; and

[0076]FIG. 8 shows a graph of NO and NO2 release from 0.083 g of 10% Apwax with 0.014 g of 10% sodium nitrite wax to give 21μ moles of NaNO₂and 25μ moles of ascorbyl palmitate. In FIG. 4 the curve with “squares”denotes NO values whereas the curve with “circles” denotes NO₂ values.

EXAMPLE 1

[0077] With reference to FIG. 1 a single colony of C albicans was usedto inoculate an overnight culture in Sabouraud's broth. 10 μl of thisbroth was added to 940 μl of a citrate/phosphate buffered Sabouraud'sbroth to which was added sodium nitrite (50 μl; final concentration 250μM) or distilled water as a control. After one hour incubation at 37°C., 10 μl was removed and cultured in 190 μl standard Sabourauds brothwith continual agitation (Gallenkamp orbital incubator) in a 96-wellmicrotitre plate at 37° C. Growth was monitored by measurement ofoptical density at 570 nm at regular time intervals. The results are amean of 16 separate experiments.

[0078] The effect of exposure to nitrite and differing hydrogen ionconcentrations on the survival of C albicans is shown in FIG. 1. Theopen bars show the growth of C albicans measured by the optical densitymethod following exposure to acid alone for 1 hour, while the closedbars show growth following exposure to acid and 250 μM sodium nitrite.There is a significant difference from the control at p>0.05(Mann-Whitney U test). It is apparent therefore that the incubation of Calbicans in acid alone for one hour had little effect on the number ofviable organisms subsequently grown, whereas in contrast the addition ofsodium nitrite at 250 μM incrementally killed C albicans as the pH wasreduced to below 4. The nitrite was in fact effective in eliminating Calbicans at pH 1 at all concentrations above 250 μM (data not shown). 5nN nitrite killed C albicans at up to pH5. It is significant that arandom sample of 10 laboratory personnel on a normal diet had fastingsalivary nitrite which varied from 23 to 220 μM (mean 114 μM) rising to409 to 1890 μM (mean 1030) 45 minutes after ingestion of 200 mgpotassium nitrate solution.

EXAMPLE 2

[0079]FIG. 2 shows growth curves of E coli following exposure to acidalone (open symbols) or acid and 250 μM nitrite (closed symbols). Growthwas significantly (p<0.05) impaired at pH 2,3 and 4 in the presence ofnitrite compared with control.

[0080] The same methods were used as in FIG. 1 except E coli (strainNCTC 10418 grown on MacConkey's agar) was used and nutrient both (OxoidCM1) was used in place of Sabouraud's broth. The results shown in FIG. 2are a mean of 20 experiments. As can be seen from FIG. 2 E coli is moresusceptible to acid than C albicans. Nevertheless exposure to pH 2 forone hour does not kill all the organisms as there is significant growthin the nutrient broth. At pH3 many more organisms survive. The additionof 250 μM nitrite to the exposure medium eliminates E coli at pH2 andsignificantly reduces the viability of this organism at pH3 and pH4.Nitrite at this concentration had no effect above pH4.

EXAMPLE 3

[0081]FIG. 3 shows growth curves of E coli following exposure to pH3 invarious nitrite concentrations (10-1000 μM final concentration). Themethods are those as for FIG. 2. FIG. 3 shows that there is a directrelationship between the toxic effects of nitrite on E coli and nitrateconcentration at pH3. Even 110 μM had a discernable effect whereas 1 mMkilled E coli completely.

EXAMPLE 4

[0082]FIG. 4 shows the generation of nitric oxide from sodium nitrite(as μM) at different acidities. Conditions were the same as those usedfor the exposure of organisms in FIG. 1. In particular nitrite was addedto citrate/phosphate buffer to achieve final concentrations shown inFIG. 4. Nitric oxide concentrations in the buffer were measured by anitric oxide sensitive meter (ISO—NO, World Precision Instruments)connected to a Maclab acquisition system and Macintosh computer.Measurements were recorded continually and readings were taken at 2minutes when nitric oxide concentration had reached a steady state. FIG.4 shows the release of nitric oxide as a result of reducing pH. Nitricoxide, which we have shown is generated under experimental conditions inFIG. 4 readily diffuses through cell membranes and has a high affinityfor iron-sulphur containing respiratory enzymes and damages bacterialDNA. When produced enzymatically by activated leucocytes, nitric acidwill kill Leishmania sp., Staphylococcus sp., Francisella sp. andMicrobacterium as well as C albicans. Reaction with superoxide underacid conditions may additionally produce highly reactive hydroxylradicals.

EXAMPLE 5

[0083] In a study to investigate the effect of a combination ofsalicylic acid at 2% w/w and sodium nitrite at 2% w/w in 9 patientvolunteers with microbiologically proven fungal infection of the feet,application of the treatment produced a microbiological cure in all butone patient after 2 weeks of therapy. The symptom score (derived from ascoring system which measures erythema, vesicles, pustules,desquamation, encrustation and pruritus) decreased from a mean of 7before treatment to a mean of 2 following treatment.

EXAMPLE 6

[0084] Investigation of the use of nitrate or nitrite administeredtopically in the mouth in the form of toothpaste, mouthwash or otherorally acceptable vehicle to reduce the number of caries-producingorganisms in dental plaque and to treat to prevent infection with Calbicans or other harmful organisms showed such application to beeffective.

[0085] The observation that oxides of nitrogen producednon-enzymatically from nitrite under conditions simulating those in thestomach kills C albicans and E coli extends these observations to theintestinal tract. E coli is closely related to Salmonella, Shigella andother pathogenic enterobacteria; all important causes of gastroenteritisin the mammal.

[0086] These results provide a rationale for active secretion of nitrateby the salivary glands. Nitrate itself is a innocuous precursor whichonly produces microbiocidal species when converted to nitrite andsubjected to acid conditions. It is possible that Lactobacilli sp.transiently produce sufficient acid in the mouth after a carbohydratemeal to control the growth of oral pathogens but clearly a moderateintake of nitrate may be a desirable prerequisite in any contaminatedenvironment despite any potential as a precursor of nitrosamines.

[0087] Further the production of intestinal nitrogen oxides may beinadequate if the oral flora which convert nitrate to nitrite aresuppressed following therapy with broad-spectrum antibiotics. Similarlyif gastric acid production is reduced, or if nitrate intake, which islargely dependent on leafy vegetables, is low this protective mechanismwill be impaired. These are precisely the situations which predispose tooral and intestinal infections.

[0088] Whereas the foregoing study has concentrated on C albicans and Ecoli and the other organisms mentioned, it may also be important forproviding protection from other serious gut pathogens which whenswallowed may cause duodenal ulceration, for example Helicobacterpylori, amoebic dysentery and chronic intestinal parasitism. Accordinglythe invention provides a dosage form for the treatment of bacterial,viral or fungal conditions, a method of sterilising an object, and acomposition therefor.

[0089] The above also suggests an inexpensive and simple means ofprevention of gastroenteritis in farmed pigs by modification of dietarynitrate intake without the use of antibiotics.

EXAMPLE 7

[0090] 32 subjects with recalcitrant viral warts were treated withvarying formulations of sodium nitrite acidified with the acidspecified. The exact formulations are given in Table 1. All 32 patientshad failed to respond to conventional topical wart applications and atleast two treatments with liquid nitrogen. 12 subjects had plantarwarts, 12 hand warts, 5 subungal or peri-ungal and 1 plane of the wartsof the hand, 1 perianal and 1 lip wart.

[0091] The warts had a duration with median 24 months this implies thatthe patients had a low chance of spontaneous improvement (See FIG. 5).No. of patients Acid Nitrite treated Salicylic 5% cream Sodium nitrite5% cream 5 Ascorbic acid 5% cream Sodium nitrite 5% cream 7 Ascorbicacid 10% cream Sodium nitrite 10% cream 2 Salicylic acid 23% in Sodiumnitrite 10% + 9 alcohol based wart paint copper acetate 0.5% Salicylicacid 23% in Sodium nitrite 10% cream 3 alcohol based wart paintSalicylic acid 23% in Sodium nitrite 15% solution 6 alcohol based wartpaint

[0092] The warts were prepared by scraping or abrading the skin toremove the dead skin then the sodium nitrite containing formulation wasapplied before applying the selected acidifying agent. The warts weretreated every night and every three days the warts were rescrapped orabraded.

[0093] Clearance of the warts occurred with a median duration of 2months regardless of the formulation of the treatment (see FIGS. 5 and6). Copper was included to catalyze the release of nitrogen oxides fromglutathione and proteins that had become nitrosated to extend therelease of nitrogen oxides.

[0094] Four treatment failures were seen; three of these in patients whowere being treated with immunosuppressive drug therapy for Lupuserthematosus, kidney transplant and dermatomyositis. Accordingly therewas an 88% cure rate in all the subjects and a 96% cure rate if theimmunosuppressed patients were excluded. Existing treatments such asusing liquid nitrogen or salicylic acid paints result in 50-80%clearance.

EXAMPLE 8

[0095] 30 patients with molluscum contagiosum lesions took part in adouble blind trial. They were randomly treated with either 5% sodiumnitrite co-applied with 5% salicylic acid under occlusion or 5% sodiumnitrite without acidification. The mean age of the subjects was 7 years(with one outlier of 47 not included in the mean). The infection had amean duration of 8.23+3.959 months. No significant difference was foundin the number of lesions per patient or the number of times treatmentwas applied in the two groups.

[0096] In the case of co-application the sodium nitrate was applied tothe skin with a cotton bud and then a fresh cotton bud was used to applythe salicylic acid. In the case of the sole application of sodiumnitrite it was applied with a cotton bud. In both cases, if possible,the area was covered with “cling-film” or Sellotape.

[0097] As seen in Table 2 in the group treated with the active treatment70% of the patients were cured and 28% of those in the control groupwere cured. The mean time to cure was 1.83+0.91 months TABLE 2 TreatmentCured Not cured Acid and Nitrite 12  4 Control  4 10

[0098] Kaplan Meier plots were performed for active and control patients(FIG. 7) and analyzed by the Logrank test which showed a significantdifference in the survival curves with cure being greater in the activegroup (p=0.0183).

EXAMPLE 9

[0099] 12 volunteers with no current or recent history of skin diseaseand taking no mediation randomly applied either low dose (0.5% nitrite)or high dose (5% nitrite) of a nitrogen oxide complex to their skin.

[0100] Subjects applied 2% w/w ascorbic acid in aqueous cream to acontrol site and an active site. Either the low dose or the high doesnitrite cream was also applied to the active site. The creams wereapplied 3 times daily at 8 hourly intervals and both the control and theactive sites were then occluded with an adhesive polythene/plasticdressing.

[0101] The last application of the cream was made 5 hours before theassessment of the reaction to allow the immediate vasodilatory effectsof the nitrogen oxide complex to subside, so measuring only residualinflammation.

[0102] The thickness of the control and active sites were measured usinga ‘Mitotoyu’ spring thickness gauge and redness was measured usingreflectance erythema metre. Two 4 mm punch biopsies were taken from theactive and control sites; one for formalin fixation for histologicalassessment, mass cell stains, neutrophil elastase and in situ nick endlabelling and the other for snap freezing and OCT embedding for theother immunohistochemical stains.

[0103] Immunohistochemical was performed using a streptavidin biotinmethod and DAB detection with the antibodies in Table 3 and usingApopTag Plus in situ nick end labelling detection kit to identifyapoptotic cells.

[0104] Staining was quantified by computerized image analysis and dataanalyzed by Wilcoxon's test for paired samples and Kruskal-Wallis' testfor non-parametric analysis of variance in the multiple independentsamples analyzed for effects of dose and duration (see Tables 4,5 and6). TABLE 3 Epitope Titre Cells Stained CD1a 0.0451388889 Langerhanscells CD3 0.555555556 pan-T cell CD4 1:150 T-helper cells CD80.555555556 T-cells suppressor/cytotoxic CD54 1:100 ICAM-1 CD60.0486111111 Macrophages CD106 1:100 VCAM-1 p53 0.0763888889 Wild typep53 protein Nitrosotyrosine* 1:100 Nitrosated tyrosine Neutrophilelastase 1:100 Neutrophils ApopTag** Manufacturers Apoptotic cellsinstructions

[0105] The reflectance erythema measurement of the nitrogen oxidecomplex treated sites was 32.25±5.46 (mean+sd) significantly higher thanthe control sites 18.08±5.81 (p=0.0022, Wilcoxon's) Skin fold thicknesswas 5.04±0.75 mm in the nitrogen oxide complex treated patches which wassignificantly greater than that of control skin 3.25±0.54 (p=0.0022,Wilcoxon's). These measures were not significantly influenced by dose orduration of exposure, except there was a trend for greater skin foldthickness in the high dose group (5.4 mm±0.21 vs 4.7 mm±0.32) (p=0.075).

[0106] Histology of all actively treated sites showed a significantincrease in oedema, endothelial swelling, cloudy swelling ofkeratinocytes, and a mixed infiltrate of lymphocytes and neutrophils.These changes were quantified on a 0-4 ordinal scale and were similar inlow does, high dose, short exposure and long exposure. The number ofmast cells seen in Azure A stained sections was similar in control andnitrogen oxide complex treated skin.

[0107] A cytotoxic effect was seen in all keratinocytes which wasmanifest as cloudy swelling. When extensive this leads to the formationof bullae high in the epidermis filled with acute inflammatory cells andcells which have undergone cytotoxic changes with constriction of thenucleus and cloudy swelling of clear cytoplasm around them. Only aminority of these degenerate cells had undergone apoptosis as judged bystaining with ApopTag. Within the viable epidermis, there was also anincrease in apoptotic cells. This suggests that normal keratinocytes,not virally infected and relatively resistant to the well knownapoptotic effects of nitric oxide. Apoptotic cells were also detected inthe dermis, particularly around adnexal structures. The positivenitrosotyrosine staining around sebaceous glands suggests that thenitrogen oxide complex was preferentially absorbed through follicles.

[0108] Nitrogen oxide complex treated skin showed significant increasesin immuno-competent cells expressing CD3, CD8, CD68 and neutrophilelastase and in the adhesion molecules which attract trafficking of thecells to the site, ICAM-1 and VCAM-1. The presence of nitrosotyrosinestaining in these cells is indicative of the formation of peroxynitrite(ONOO—) and of p53 which indicates that part of the effect of thecomplex is mediated through toxicity towards DNA in these cells. Inhealthy skin nitrogen oxide complex did cause some apoptosis but thiswas surprisingly small at the doses used and we postulate that theeffect is likely to be in infected cells. The antigen processing cellsof the skin (CD1a positive) were seen to lose dendricity and drop fromthe epidermis so there were significantly fewer in the treated skin. Asthese cells behave in this way when activated and functioning to processa newly recognized antigen, this would seem to offer further evidencefor an immunopotentiating role for the nitrogen oxide complex. Ki-67staining for dividing cells did not differ in control or active sites.This would suggest that in warts, for example, the action is not one ofreducing cell proliferation. Kruskal-Walls test was used to test theeffects of time or duration of nitrogen oxide complex treatment onclinical and immunohistochemical response. The effect of the dosage onthe skins reaction is given in Table 5. There were fewer CD4 positivecells in the high dose than the low dose group, and likewise with CD68positive cells. Although Ki-67 positive cells were not significantlydifferent between the control site and the nitrogen oxide complextreated site, there was a significant increase with high dose comparedwith low dose.

[0109] After 24 and 48 hours exposure to the nitrogen oxide complex theextent of apoptosis was measured, see Table 6. There was significantlygreater apoptosis after 48 hours than after 24 hours. The CD4 positivecells count rose significantly after 48 hours compared to after 12hours. The difference for p53 was not quite statistically significant.Similarly, cloudy swelling tended to be greater in the longer durationtreatment but was not statistically significant. TABLE 4 NitrogenSignificance Oxide Complex Control Wilcoxon's Mean S.D. Mean S.D. TestApopTag 12.5 10.1 0.41 1.6 0.0033 Ki67 6.82 3.82 6.62 2.854 0.67  CD1a*0.86 0.69 3.43 0.53 0.022  CD3 574.7 396.3 216.1 122.1 0.0186 CD4 608.2458.2 176.3 149.9 0.0125 CD8 275.7 193.1 122.1 106.1 0.0284 CD68 673.1542.4 301.4 361.3 0.0044 Nitro-sotyrosine* 3.4 0.7 0.9 1.1 0.043  p53214.4 266.4 22.08 53.8 0.0029 Neutrophils 569.4 385.9 71 113.1 0.043 ICAM-1 705.9 704.5 201.9 160.9 0.0209 VCAM-1 1.5 1.17 0.5 0.9 0.0357

[0110] Ki67 was counted in the epidermis and ApopTag positive cellscounted per standard section through a 3 mm punch biopsy. All othercounts were done by computerized image analysis on a fixed standardmeasuring frame and are expressed as cells per mm 2. TABLE 5 High DoseLow Dose (cells/mm2) (cells/mm2) Kruskal- Mean S.D. Mean S.D. WallisTest Ki67 152   35.2  73.6  8  0.01 CD4 280   78.4 936   185.6 0.02 CD68379.2 65.6 916.8 262.4 0.04

[0111] TABLE 6 12/24* hrs 48 hrs Kruskal- Mean S.D. Mean S.D. WallisTest ApopTag*  3.5  1.82  14.16  2.56 <0.005 CD4 285.9 193.6  824  193.6    0.05  Cloudy swelling**  1.7  0.33  2.5  0.224   0.07  p53 70.1  47.04 335.2 125.7    0.07 

[0112] The nitrogen oxide complexes of the invention may be formed by acombination of ascorbic acid and nitrite on the skin, which causes therelease of nitrogen oxides, inter alia nitric oxide, nitrous oxide,nitrogen dioxide and dinitrogen trioxide. The increase in T helper cellsand macrophages was greater in low dose subjects and suggests that atlower doses nitrogen oxides can be pro-inflammatory but at higher dosesbecomes cytotoxic to the immunocompetent cells and begins to exert aninhibitory effect. The nitrogen oxide complex led to a marked inductionof ICAM-1 and a moderate increase in VCAM-1 expression. The pattern ofinflammation was unusual in showing a marked infiltrate of macrophagesafter only 24 hours, so showing that activated macrophages use nitrogenoxides to specifically attract more macrophages to kill a pathogen.

[0113] The promotion of apotosis and recruitment of all theimmunocompetent cells required for effective recognition of a pathogenby the immune system of a host, results from application of apreparation of a combination of nitrite or precursor of nitrogen oxidesand an acidifying agent. Accordingly, these findings support a potentialimmunopotentiating effect of the combination of nitrite or otherprecursor of nitrogen oxides such as NO or NO₂ and a acidifying agent.

EXAMPLE 10

[0114] A two part component delivery system was made up. Each componentwas in the form of a wax stick which can be rubbed onto an effectivearea at regular intervals in accordance with a physician's instructions.

[0115] The two components were made up as follows:

[0116] 10% ASCORBYL PALMITATE Component Ascorbyl Palmitate    10% WhiteSoft Paraffin 25 Light Liquid Paraffin 20 Hard Paraffin 20 Arlacel 16515 Cetosteryl Alcohol 10

[0117] Method

[0118] 1. Weigh all the components into a vessel.

[0119] 2. Heat the vessel and stir the mixture until all the componentshave melted and the mixture is homogenous.

[0120] 3. Pour the molten wax into jars and allow to cool to roomtemperature.

[0121] 10% SODIUM NITRITE WAX Components Phase A Light Liquid Paraffin7.5% White Soft Paraffin 20 Arlacel 582 10 Cetosteryl alcohol 10Phenoxyethanol 1 Phase B Sodium Nitrite 10 Purified Water 20

[0122] Method

[0123] 1. Weigh the Phase A components into a vessel, heat to 70° C. andstir until homogenous.

[0124] 2. Weigh the Phase B components into another vessel heat to 70°C. and stir, ensure that all the sodium nitrite has dissolved.

[0125] 3. When both phases have reach 70° C., add phase A to phase B andhomogenize for 5 minutes.

[0126] 4. Pour the molten wax into jars and allow to cool to roomtemperature.

[0127] As is shown from FIG. 8, the use of this admixture tends torelease a substantial excess of NO from the two-part delivery system.This is possibly because NO is a small molecule which results in a moreeffective treatment of viral skin diseases.

What we claim is:
 1. A dosage form for the treatment of bacterial, viralor fungal conditions which comprises, a pharmaceutically acceptableacidifying agent in an amount sufficient to reduce the pH at anenvironment of use to below pH4, and a pharmaceutically acceptablesource of nitrite ions or a nitrate precursor therefor; wherein saidacidifying agent and said source of nitrite ions or nitrate precursorare separately disposed in respective pharmaceutically acceptablecarriers for admixture at the intended environment of use to release NOor NO₂ ions.
 2. A dosage form according to claim 1 wherein theacidifying agent is an acid or an acid salt.
 3. A dosage form accordingto claim 1 wherein the pharmaceutically acceptable carriers are selectedfrom inert creams, ointments, tablets or are in liquid form.
 4. A dosageform according to claim 1 wherein the condition to be treated is causedby an organism selected from the group consisting of Albicans sp.,Leishmania sp., Staphylococcus sp., Francisella sp., Microbacterium, E.coli Tinea pedis, Heliobacter pylorii and amoebic dysentery.
 5. A dosageform according to claim 1 used to treat a fungal infection of the feet.6. A dosage form according to claim 1 wherein the condition fortreatment is virus mediated.
 7. A dosage form adapted for the treatmentof a virus mediated condition by topical application of a medicament toor adjacent to an environment of use, comprising an admixture ofnitrogen oxides generated at the environment of use from apharmaceutically acceptable acidifying agent in an amount sufficient toreduce the pH or the environment of use to below pH, and apharmaceutically acceptable source of nitrogen oxides or a nitrateprecursor therefor.
 8. A dosage form according to claim 7 where theacidifying agent is also a reducing agent.
 9. A dosage form according toclaim 7 when the acidifying agent is present in an amount sufficient toreduce the pH at the environment of use to a value above pH2.
 10. Adosage form according to claim 7 wherein the nitrogen oxides are derivedfrom an inorganic nitrite.
 11. A dosage form according to claim 7 wherethe pharmaceutically acceptable acidifying agent is selected fromabsorbic and, ascorbyl palmitate, salicylic acid, lactic acid, citricacid, benzoic acid and tartaric acid.
 12. A dosage form according toclaim 7 wherein the source of nitrogen acids is an alkali metal nitriteor precursor therefor and constitutes 0.5 to 30% by weight of the totaldosage form.
 13. A dosage form according to claim 7 wherein the viruscondition is engendered by molluscum contagiosum, herpes simplex types 1& 2, valicella zoster virus, and papilloma virus
 14. A delivery systemfor the topical application of a medicament for the in vivo treatment ofthe epidermis, comprising an adhesive layer and a support layerimpregnated with at least one of the components of the medicamentcharacterized in that the components of the medicament compriseseparately a pharmaceutically acceptable acidifying agent and apharmaceutically acceptable source of nitrogen oxides or a precursortherefor, and a means for combining the pharmaceutically acceptableacidifying agent with the source of nitrogen oxides at the environmentof use.
 15. A delivery system according to claim 14 wherein the deliverysystem comprises microspheres.
 16. A delivery system according to claim14 wherein the delivery system comprises liposomes.
 17. A two-partdelivery system for topical application of a medicament for the in vivotreatment of a viral, bacterial or fungal infection of the epidermis,said system comprising separately; (a) a first waxy component comprisinga pharmaceutically acceptable acidifying agent; and (b) a second waxycomponent comprising a pharmaceutically acceptable source of nitrogenoxides, whereby when topically applied simultaneously or immediatelysequentially to an environment of use, active nitrogen oxides arereleased thereto.
 18. A delivery system according to claim 17 whereinthe acidifying agent is a reducing organic acid or salt.